The present invention relates to a method of virus-filtering a solution that contains at least one macromolecule, by virtue of the total salt content of the solution lying in the range of from about 0.2 M up to saturation of the solution with the salt concerned. The inventive method reduces the residence time and the extent to which the solution need to be diluted, and optimizes the yield when virus-filtering primarily proteins, polysaccharides and polypeptides. The reduction in virus content is at least as good as with conventional techniques where the total salt content is low. The present invention facilitates virus filtration with the aid of the so-called xe2x80x9cdead-endxe2x80x9d technique, which affords several process and economic advantages in comparison with the tangential virus-filtering technique normally used. When virus-filtering the plasma protein factor IX, the yield obtained in the virus-filtering stage is increased from about 70% to above 95%, by raising the salt content of the solution in accordance with the present invention.
The problem of virus contamination of various protein preparations intended for the medication of human beings has received greater notice in recent years. For instance, occasional reports have been submitted concerning, e.g., blood proteins that have been contaminated with hepatitis virus A, hepatitis virus B, hepatitis virus C and/or Human Immunodeficiency Virus (HIV). In keeping with these reports, the authorities of several countries have sharpened their requirements with regard to cleansing protein preparations of their possible virus contaminants.
In present-day, conventional techniques, viruses are inactivated with the aid of chemical additives, primarily solvents and detergents, and/or by exposing the viruses to elevated temperatures. The former method has the drawback of functioning solely on virus with lipid envelopes, for instance hepatitis virus B and HIV. The latter technique mentioned above has the drawback that many proteins are thermally unstable at those temperatures required to effectively reduce the contaminating virus.
U.S. Pat. No. 4,473,494 (assigned to the U.S. Secretary of the Army) discloses a method for production of stroma-free, non-heme protein-free hemoglobin by use of zinc ions to promote precipitation of a zinc ion-bound insoluble hemoglobin complex, followed by membrane ultrafiltration of the zinc-hemoglobin complex from the filtrate fluid medium. In the only step where viruses are said to be removed from hemoglobin, the total salt content is below 0.05 M, i.e. the total content of salt is conventional.
EP-A-0307373 (assigned to Ares-Serono) relates to removal of viruses and/or other contaminants from biological materials in fluid form by using ultrafiltration membranes having a 100,000 Da cut-off. A preferred biological material is human growth hormone. In the examples of EP-A-0307373, the total content of salt in the virus-filtering step lies in the range of from 0.01 up to 0.10 M (NH4CO3), i.e. the total content of salt is conventional.
There is thus a need for an effective virus-reducing method which can be applied to different types of macromolecules, primarily proteins, and on different types of viruses.
One object of the present invention is to markedly reduce the residence time when virus-filtering solutions that contain macromolecules.
Another object of the present invention is to markedly reduce the liquid volumes when virus-filtering solutions that contain macromolecules.
A further object of the present invention is to reduce the filter area required to effectively virus-filter solutions that contain macromolecules.
Yet another object of the present invention is to achieve a macromolecule yield in excess of about 90% in the virus-filtering stage.
Still another object of the present invention is to reduce the polymerization obtained on the virus filter surface, so as to enable the rate of flow to be increased and the process time to be decreased.
These and other objects are fulfilled by the present invention, which relates to a method of virus-filtering a solution containing at least one macromolecule wherein the total salt content of the solution lies within the range of from about 0.2 M up to saturation of the solution with the salt concerned.
The inventor of this invention has thus found that virus filtration can be effected much more effectively than previously known, by increasing the salt content of the solution. This discovery is surprising, because hitherto in virus filtration of proteins it has been believed that solely the protein concentration, the rate of flow and the pH have had any influence on the process.
It is believed that the enhanced filtering effect achieved at higher salt concentrations is because the protein contracts and can therewith pass more easily through the filter pores. It is also conceivable, that the interaction is reduced between macromolecules themselves and/or between the macromolecules and the material of the filter membrane. It is also conceivable that proteins having a large number of hydrophobic groups are influenced to a greater extent by an elevated salt concentration.
The closer the molecular weight, or relative molecular mass, of the macromolecule lies to the pore size of the filter membrane, the more effective the present invention. The effectiveness of the present invention is also enhanced when the difference in the size and/or the molecular weight of the contaminants and the product increases, i.e. with increasing concentrations of high molecular contaminants in the product.
The present invention also facilitates specific fractions to be separated from a desired product, for instance enables undesirable proteins to be separated from the protein that constitutes the product.
The use of a high salt content according to the present invention, also enables the use of the socalled xe2x80x9cdead-endxe2x80x9d filtering technique. This preferred embodiment, has several advantages over conventional tangential filtering processes normally applied, especially with a pore size of about 5-30 nm. For instance, the equipment and operating procedures required are much simpler and therewith less expensive. The use of xe2x80x9cdead-endxe2x80x9d filtration also reduces the loss of the macromolecule, reduces the process time, increases the permeability of the macromolecule through the filter, and also enables a generally constant concentration of the macromolecule to be achieved over the filter as well as a constant membrane pressure. Another advantage with the dead-end filtering technique, is the fact that scaling-up of virus filtering processes from laboratory to industrial scale is considerably facilitated.
When practicing the present invention, the total salt content of the solution suitably lies within the range of from 0.3 up to 3.0 M, preferably within the range of from 0.4 up to 2.5 M, and more preferably within the range of from 0.6 up to 2.0 M. It is particularly preferred that the total salt content of the solution lies within the range of from 0.8 up to 1.5M.
When necessary, the total salt content of the solution can be adjusted by adding any acceptable salt. For instance, it is possible to use soluble inorganic salts, soluble organic salts or combinations of such salts. It is assumed that important process advantages are obtained when using salts which exhibit a high salting-out effect in accordance with the so-called Hofmeister series. Reference is here made to S. Glasstone, Textbook of Physical Chemistry, van Nostrand Co., Toronto, 2nd edition, April 1946, pp. 1254-1259. The most important examples of anions which have such high salting-out effect are citrate, tartrate, sulfate, acetate and phosphate. Cations that can be used advantageously when practicing the present invention are monovalent cations, such as sodium, potassium and ammonium, as well as divalent cations, such as calcium. Sodium chloride, potassium chloride, sodium acetate and sodium citrate or combinations thereof are particularly preferred salts in accordance with the invention, in view of the advantages that are afforded by pharmaceutically acceptable additives. It is also conceivable to add one or more salts in sequence, when the filtration process is carried out in two or more steps.
A protein concentration within the range of from about 5 up to about 10 mg/ml solution is often recommended for virus filtration. When applying the present invention, it was surprisingly found that solutions having a higher protein concentration, from about 10 up to about 20 mg/ml, could be processed advantageously through the virus filter.
The solution should have a temperature within the range from 0xc2x0 C. up to the temperature at which the protein concerned is denatured. The temperature of the solution suitably lies within the range of from 10xc2x0 C. up to 50xc2x0 C., preferably from 20xc2x0 C. up to 35xc2x0 C.
When practicing the present invention, the solution should have a pH in the range of from about 3 up to about 9, suitably from 4 up to 8. The pH of the protein solution should not lie too close to the isoelectric point of the protein concerned. For instance, in the case of gammaglobulin, a better result is obtained with a pH of 5.5 than with a pH of 6.8.
In the present invention, solution refers to a solution that contains at least 50 percent by weight of water, optionally including one or more solvents, such as methanol, ethanol, aacetone or acetonitrile.
The present invention can be used to optimize process procedures when virus-filtering solutions that contain a large number of different types of macromolecules. Examples of such molecules are proteins, polysaccharides and polypeptides or combinations thereof. The origin of the macromolecules is irrelevant to the use of the present invention. The macromolecules may thus derive from the plant kingdom or the animal kingdom or may be produced initially by industrial processes. However, the macromolecules are suitably of human or animal origin or engineered genetically (recombinants).
Particularly appropriate proteins in regard of the present invention are factor VIII, factor IX, antithrombin III, gammaglobulin, albumin, streptokinase, apolipoproteins and growth hormones.
A particularly preferred factor IX product is Nanotiv(copyright), which is supplied by Pharmacia AB, Stockholm, Sweden. The advantage with this product is that its specific activity prior to filtration is sufficiently high to enable the use of a filter of very fine structure. This enables the virus concentration to be lowered to an extremely low level, at the same time as the filtering process itself is very rapid and produces a high yield.
Preferred types of factor VIII are deletion derivatives of recombinant produced factor VIII products. A particularly preferred factor VIII product is r-VIII SQ supplied by Pharmacia AB, Stockholm, Sweden. One advantage with this product is that the recombinant produced product molecule lacks the inactive intermediate part of the natural factor VIII molecule. This gives the molecule a mean molecular weight of about 170,000. A molecule of this size is particularly suited for filtration with such filters as those which enable a considerable virus reduction to be achieved.
Preferred apolipoproteins include apolipoprotein AI (Apo AI), apolipoprotein AII (Apo AII), apolipoprotein AIV (Apo AIV), apolipoprotein E (Apo E) and variants or mixtures thereof. Variants include preforms, fragments and truncated, extended or mutated forms of Apo AI, Apo II, Apo IV and Apo E. Mutated forms in which at least one arginine group has been replaced with a cystein group are particularly preferred. One such mutated form is Apo A-IMilano (Apo A-IM), also produced with recombinant DNA technique by Pharmacia AB, Stockholm, Sweden.
Polysaccharides which are particularly preferred in accordance with the present invention are glycosaminoglycans and bacteria polysaccharides. Examples of glycos-aminoglycans are heparins, heparin fragments, heparin derivatives, heparan sulfate and hyaluronic acid. A particularly preferred group of glycosaminoglycans is comprised of low molecular weight heparins having a mean molecular weight of up to about 10,000, preferably from 2,000 up to 8,000.
According to the present invention, particularly suitable polypeptides are bioactive polypeptides, such as recombinant human growth hormones produced in mammalian cells.
The present invention can thus be used to optimize the process of virus-filtering solutions that contain, e.g., proteins, polysaccharides and polypeptides. However, the invention is described in the following with reference to solutions that contain proteins, more specifically proteins that occur naturally in the human organism.
Those viruses that may be present in protein solutions will normally be much larger than the proteins themselves. It is thus presumable that viruses can be removed from proteins in accordance with size, for instance by filtration.
Viruses that can be removed efficiently with the present invention, can have a size smaller than about 350 nm. The size of the viruses that can be removed, suitably is smaller than 200 nm, preferably smaller than 150 mn. Normally, the viruses that can be removed are larger than about 20 nm, i.e. the approximate size of the parvo virus.
The present invention is primarily intended for removing viruses from macromolecules, where the macromolecules are the product of interest. It is, however, within the scope of the invention, to use the present method for separating viruses from macromolecules, where the viruses are the product of interest. An example, is the purification of parvovirus for use as a testing agent, and poliovirus for use a vaccine, wherein e.g. proteins and polysaccharides can be removed by the present method.
Virus filtration is normally carried out in a tangential filtering process or in a so-called xe2x80x9cdead-endxe2x80x9d filtering process. In tangential virus filtration, the protein solution is pumped around at a constant rate of flow on the retention side, while another pump draws the protein solution through the filter by suction. When a given volume has been obtained on the retention side, a buffer is added on the retention side. This procedure is repeated a number of times, as necessary, with the major part of the remaining protein passing through the filter while retaining the virus on the retention side. Such a process is called diafiltration. The filter is normally discarded after each run, to avoid transferring the virus.
In the case of socalled xe2x80x9cdead-endxe2x80x9d virus filtration, the same virus filter as that used in tangential virus filtration can be used, although the peripheral equipment and operating procedures are much simpler and less expensive than in the case of tangential virus filtration. Thus, in principle, xe2x80x9cdead-endxe2x80x9d filtration involves placing the macromolecule-containing solution in a pressure vessel prior to filtration and pressing the solution through the virus filter with the aid of a pressure source, suitably nitrogen (gas).
The degree of fineness of filters generally, is normally given as pore size or the approximate molecular weight (relative molecular mass) at which the molecules are stopped by the filter, the so called cut-off. In the present invention, the virus filters can have a cut-off of about 1,000,000, suitably 500,000. To remove small viruses, the virus filters should have a cut-off of 200,000, preferably 100,000. To reach a maximum virus-reduction, the virus filter should have a cut-off slightly higher than the macromolecule which is virus-filtered.
Virus filters are known in the art and are supplied by Millipore from Massachusetts, USA and Asahi Chemical Industry Co., Ltd. from Japan, among others. Millipore supplies filters having two different types of membrane, depending on the size of the protein concerned. For instance, Millipore supplies, among others, Viresolve(trademark)/70 for proteins having a molecular weight, or relative molecular mass, of up to about 70,000, and Viresolve(trademark)/180 for proteins having a molecular weight of up to about 180,000. This latter filter can be used for monoclonal antibodies, for instance. Asahi Chemical Industry supplies, among other things, Planova(trademark) 35 and Planova(trademark) 15 filters, this latter filter being used to remove smaller viruses, such as the polio virus.
As mentioned before, the choice of filter will depend on the size of the protein concerned, among other things. Factor IX, antithrombin III, human serum albumin (HSA) and Apo A-IM (the dimer) all have a molecular weight of roughly 60,000-70,000, wherein Viresolve(trademark)/70, for instance, is a suitable alternative. Gammaglobulin has a molecular weight of about 180,000, wherein Viresolve(trademark)/180, for instance, is a suitable alternative. The latter filter is also suitable for use with the recombinant produced factor VIII product, r-VHIII SQ, which has a molecular weight of about 170,000, as mentioned before.
The possibility of choosing a fine structure filter also assumes that the protein solution has a high degree of purity prior to filtration. In turn, the use of a fine structure filter is a prerequisite for the ability to produce protein solutions which have a very low virus content in the end product. Thus, in order to be able to reduce the virus concentration to a very low level, there is required a filter of very fine structure, for instance Viresolve(trademark)/70. The virus concentration cannot be lowered to quite such a low level when using Viresolve(trademark)/180.
The effectiveness, or efficiency, of the filtering stage is influenced by the purity of the protein solution delivered to the filter. In this regard, a high specific activity prior to filtration results in a higher yield in the filtering stage. For instance, in the case of preferred embodiments applied when filtering solutions that contain factor IX, it has been found that the protein yield in the filtering stage can be increased from about 70% to above 95%. However, when practicing the present invention, it is possible to achieve protein yields of above 90%, even when working with solutions of low specific activity.
With the present invention, it is possible to reduce the content of very small non-enveloped viruses, such as the parvovirus, by at least 3 logs, suitably at least 4 logs, and preferably at least 5 logs. The reduction is very good with the tangential technique, but even better with the xe2x80x9cdead-endxe2x80x9d technique, when applied according to the present invention.
According to the invention, virus filtration is preferably carried out at the end of a protein manufacturing sequence, since a high specific activity prior to filtration will result in a higher protein yield in the filtering stage. The present invention is preferably applied as a last purification stage, optionally followed by a stage for adjusting, for instance, protein concentration, salt content or the pH of the end product. A following diafiltration stage using a UF-membrane may also be applied to remove salts which although advantageous from a process or economic aspect during virus filtration should not be included in the end product. Protein solutions which are ready for administration will normally contain a physiological solution, for instance 0.15 M sodium chloride at a pH of 7, in combination with one or more stabilizers, such as saccharose or amino acids. The virus filtration process may also be carried out in two or more steps, with or without intermediate process steps.
The present invention effectively reduces the content of virus with lipid envelopes and viruses without lipid envelopes. Examples of viruses without a lipid envelope are the hepatitis virus A, polio virus and parvo virus, which, are relatively small viruses. Examples of viruses with a lipid envelope are the hepatitis virus B, the hepatitis virus C and the Human Immunodeficiency Virus (HIV).
The invention will now be illustrated in more detail with the aid of exemplifying, non-limiting examples.
Experiments were carried out in which the sieving coefficient of proteins, or protein permeability factor, was first determined at different filtrate flowrates. The sieving coefficient, or protein permeability factor, is given as P/R, where P is the concentration of protein on the permeate side (the filtrate side) measured by absorption at 280 nm (A280) and R is the concentration of protein on the retention side (R) measured by absorption at 280 nm (A280). The filtrate flowrate which gave the highest sieving coefficient in the absence of polymerization on the filter was then chosen. A yield optimization was also made with some macromolecules.